Tag Archives: genetic mutations

CRISPR-Cas12a as a new diagnostic tool

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Similar to Cas9, Cas12a is has an added feature as noted in this February 15, 2018 news item on ScienceDaily,

Utilizing an unsuspected activity of the CRISPR-Cas12a protein, researchers created a simple diagnostic system called DETECTR to analyze cells, blood, saliva, urine and stool to detect genetic mutations, cancer and antibiotic resistance and also diagnose bacterial and viral infections. The scientists discovered that when Cas12a binds its double-stranded DNA target, it indiscriminately chews up all single-stranded DNA. They then created reporter molecules attached to single-stranded DNA to signal when Cas12a finds its target.

A February 15, 2018 University of California at Berkeley (UC Berkeley) news release by Robert Sanders and which originated the news item, provides more detail and history,

CRISPR-Cas12a, one of the DNA-cutting proteins revolutionizing biology today, has an unexpected side effect that makes it an ideal enzyme for simple, rapid and accurate disease diagnostics.

blood in test tube

(iStock)

Cas12a, discovered in 2015 and originally called Cpf1, is like the well-known Cas9 protein that UC Berkeley’s Jennifer Doudna and colleague Emmanuelle Charpentier turned into a powerful gene-editing tool in 2012.

CRISPR-Cas9 has supercharged biological research in a mere six years, speeding up exploration of the causes of disease and sparking many potential new therapies. Cas12a was a major addition to the gene-cutting toolbox, able to cut double-stranded DNA at places that Cas9 can’t, and, because it leaves ragged edges, perhaps easier to use when inserting a new gene at the DNA cut.

But co-first authors Janice Chen, Enbo Ma and Lucas Harrington in Doudna’s lab discovered that when Cas12a binds and cuts a targeted double-stranded DNA sequence, it unexpectedly unleashes indiscriminate cutting of all single-stranded DNA in a test tube.

Most of the DNA in a cell is in the form of a double-stranded helix, so this is not necessarily a problem for gene-editing applications. But it does allow researchers to use a single-stranded “reporter” molecule with the CRISPR-Cas12a protein, which produces an unambiguous fluorescent signal when Cas12a has found its target.

“We continue to be fascinated by the functions of bacterial CRISPR systems and how mechanistic understanding leads to opportunities for new technologies,” said Doudna, a professor of molecular and cell biology and of chemistry and a Howard Hughes Medical Institute investigator.

DETECTR diagnostics

The new DETECTR system based on CRISPR-Cas12a can analyze cells, blood, saliva, urine and stool to detect genetic mutations, cancer and antibiotic resistance as well as diagnose bacterial and viral infections. Target DNA is amplified by RPA to make it easier for Cas12a to find it and bind, unleashing indiscriminate cutting of single-stranded DNA, including DNA attached to a fluorescent marker (gold star) that tells researchers that Cas12a has found its target.

The UC Berkeley researchers, along with their colleagues at UC San Francisco, will publish their findings Feb. 15 [2018] via the journal Science’s fast-track service, First Release.

The researchers developed a diagnostic system they dubbed the DNA Endonuclease Targeted CRISPR Trans Reporter, or DETECTR, for quick and easy point-of-care detection of even small amounts of DNA in clinical samples. It involves adding all reagents in a single reaction: CRISPR-Cas12a and its RNA targeting sequence (guide RNA), fluorescent reporter molecule and an isothermal amplification system called recombinase polymerase amplification (RPA), which is similar to polymerase chain reaction (PCR). When warmed to body temperature, RPA rapidly multiplies the number of copies of the target DNA, boosting the chances Cas12a will find one of them, bind and unleash single-strand DNA cutting, resulting in a fluorescent readout.

The UC Berkeley researchers tested this strategy using patient samples containing human papilloma virus (HPV), in collaboration with Joel Palefsky’s lab at UC San Francisco. Using DETECTR, they were able to demonstrate accurate detection of the “high-risk” HPV types 16 and 18 in samples infected with many different HPV types.

“This protein works as a robust tool to detect DNA from a variety of sources,” Chen said. “We want to push the limits of the technology, which is potentially applicable in any point-of-care diagnostic situation where there is a DNA component, including cancer and infectious disease.”

The indiscriminate cutting of all single-stranded DNA, which the researchers discovered holds true for all related Cas12 molecules, but not Cas9, may have unwanted effects in genome editing applications, but more research is needed on this topic, Chen said. During the transcription of genes, for example, the cell briefly creates single strands of DNA that could accidentally be cut by Cas12a.

The activity of the Cas12 proteins is similar to that of another family of CRISPR enzymes, Cas13a, which chew up RNA after binding to a target RNA sequence. Various teams, including Doudna’s, are developing diagnostic tests using Cas13a that could, for example, detect the RNA genome of HIV.

infographic about DETECTR system

(Infographic by the Howard Hughes Medical Institute)

These new tools have been repurposed from their original role in microbes where they serve as adaptive immune systems to fend off viral infections. In these bacteria, Cas proteins store records of past infections and use these “memories” to identify harmful DNA during infections. Cas12a, the protein used in this study, then cuts the invading DNA, saving the bacteria from being taken over by the virus.

The chance discovery of Cas12a’s unusual behavior highlights the importance of basic research, Chen said, since it came from a basic curiosity about the mechanism Cas12a uses to cleave double-stranded DNA.

“It’s cool that, by going after the question of the cleavage mechanism of this protein, we uncovered what we think is a very powerful technology useful in an array of applications,” Chen said.

Here’s a link to and a citation for the paper,

CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity by Janice S. Chen, Enbo Ma, Lucas B. Harrington, Maria Da Costa, Xinran Tian, Joel M. Palefsky, Jennifer A. Doudna. Science 15 Feb 2018: eaar6245 DOI: 10.1126/science.aar6245

This paper is behind a paywall.

New nanomapping technology: CRISPR-CAS9 as a programmable nanoparticle

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A November 21, 2017 news item on Nanowerk describes a rather extraordinary (to me, anyway) approach to using CRRISP ( Clustered Regularly Interspaced Short Palindromic Repeats)-CAS9 (Note: A link has been removed),

A team of scientists led by Virginia Commonwealth University physicist Jason Reed, Ph.D., have developed new nanomapping technology that could transform the way disease-causing genetic mutations are diagnosed and discovered. Described in a study published today [November 21, 2017] in the journal Nature Communications (“DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle”), this novel approach uses high-speed atomic force microscopy (AFM) combined with a CRISPR-based chemical barcoding technique to map DNA nearly as accurately as DNA sequencing while processing large sections of the genome at a much faster rate. What’s more–the technology can be powered by parts found in your run-of-the-mill DVD player.

A November 21, 2017 Virginia Commonwealth University news release by John Wallace, which originated the news item, provides more detail,

The human genome is made up of billions of DNA base pairs. Unraveled, it stretches to a length of nearly six feet long. When cells divide, they must make a copy of their DNA for the new cell. However, sometimes various sections of the DNA are copied incorrectly or pasted together at the wrong location, leading to genetic mutations that cause diseases such as cancer. DNA sequencing is so precise that it can analyze individual base pairs of DNA. But in order to analyze large sections of the genome to find genetic mutations, technicians must determine millions of tiny sequences and then piece them together with computer software. In contrast, biomedical imaging techniques such as fluorescence in situ hybridization, known as FISH, can only analyze DNA at a resolution of several hundred thousand base pairs.

Reed’s new high-speed AFM method can map DNA to a resolution of tens of base pairs while creating images up to a million base pairs in size. And it does it using a fraction of the amount of specimen required for DNA sequencing.

“DNA sequencing is a powerful tool, but it is still quite expensive and has several technological and functional limitations that make it difficult to map large areas of the genome efficiently and accurately,” said Reed, principal investigator on the study. Reed is a member of the Cancer Molecular Genetics research program at VCU Massey Cancer Center and an associate professor in the Department of Physics in the College of Humanities and Sciences.

“Our approach bridges the gap between DNA sequencing and other physical mapping techniques that lack resolution,” he said. “It can be used as a stand-alone method or it can complement DNA sequencing by reducing complexity and error when piecing together the small bits of genome analyzed during the sequencing process.”

IBM scientists made headlines in 1989 when they developed AFM technology and used a related technique to rearrange molecules at the atomic level to spell out “IBM.” AFM achieves this level of detail by using a microscopic stylus — similar to a needle on a record player — that barely makes contact with the surface of the material being studied. The interaction between the stylus and the molecules creates the image. However, traditional AFM is too slow for medical applications and so it is primarily used by engineers in materials science.

“Our device works in the same fashion as AFM but we move the sample past the stylus at a much greater velocity and use optical instruments to detect the interaction between the stylus and the molecules. We can achieve the same level of detail as traditional AFM but can process material more than a thousand times faster,” said Reed, whose team proved the technology can be mainstreamed by using optical equipment found in DVD players. “High-speed AFM is ideally suited for some medical applications as it can process materials quickly and provide hundreds of times more resolution than comparable imaging methods.”

Increasing the speed of AFM was just one hurdle Reed and his colleagues had to overcome. In order to actually identify genetic mutations in DNA, they had to develop a way to place markers or labels on the surface of the DNA molecules so they could recognize patterns and irregularities. An ingenious chemical barcoding solution was developed using a form of CRISPR technology.

CRISPR has made a lot of headlines recently in regard to gene editing. CRISPR is an enzyme that scientists have been able to “program” using targeting RNA in order to cut DNA at precise locations that the cell then repairs on its own. Reed’s team altered the chemical reaction conditions of the CRISPR enzyme so that it only sticks to the DNA and does not actually cut it.

“Because the CRISPR enzyme is a protein that’s physically bigger than the DNA molecule, it’s perfect for this barcoding application,” Reed said. “We were amazed to discover this method is nearly 90 percent efficient at bonding to the DNA molecules. And because it’s easy to see the CRISPR proteins, you can spot genetic mutations among the patterns in DNA.”

To demonstrate the technique’s effectiveness, the researchers mapped genetic translocations present in lymph node biopsies of lymphoma patients. Translocations occur when one section of the DNA gets copied and pasted to the wrong place in the genome. They are especially prevalent in blood cancers such as lymphoma but occur in other cancers as well.

While there are many potential uses for this technology, Reed and his team are focusing on medical applications. They are currently developing software based on existing algorithms that can analyze patterns in sections of DNA up to and over a million base pairs in size. Once completed, it would not be hard to imagine this shoebox-sized instrument in pathology labs assisting in the diagnosis and treatment of diseases linked to genetic mutations.

Here’s a link to and a citation for the paper,

DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle by Andrey Mikheikin, Anita Olsen, Kevin Leslie, Freddie Russell-Pavier, Andrew Yacoot, Loren Picco, Oliver Payton, Amir Toor, Alden Chesney, James K. Gimzewski, Bud Mishra, & Jason Reed. Nature Communications 8, Article number: 1665 (2017) doi:10.1038/s41467-017-01891-9 Published online: 21 November 2017

This paper is open access.